Domain swapping is a mechanism of protein oligomerization by which two or more subunits exchange structural elements to generate an intertwined complex. Numerous studies support a diversity of swapping mechanisms in which structural elements can be exchanged at different stages of the folding pathway of a subunit. Here, we used single-molecule optical tweezers technique to analyze the swapping mechanism of the forkhead DNA-binding domain of human transcription factor FoxP1. FoxP1 populates folded monomers in equilibrium with a swapped dimer. We generated a fusion protein linking two FoxP1 domains in tandem to obtain repetitive mechanical folding and unfolding trajectories. Thus, by stretching the same molecule several times, we detected either the independent folding of each domain or the elusive swapping step between domains. We found that a swapped dimer can be formed directly from fully or mostly folded monomer. In this situation, the interaction between the monomers in route to the domain-swapped dimer is the rate-limiting step. This approach is a useful strategy to test the different proposed domain swapping mechanisms for proteins with relevant physiological functions.

域交换是蛋白质寡聚化的一种机制，通过该机制，两个或多个亚基交换结构元件以产生相互交织的复合物。许多研究支持多种交换机制，其中结构元素可以在亚基折叠途径的不同阶段进行交换。在这里，我们使用单分子光镊技术分析了人类转录因子FoxP1叉头DNA结合域的交换机制。FoxP1 填充折叠的单体与交换的二聚体平衡。我们生成了一个融合蛋白，将两个 FoxP1 结构域串联起来，以获得重复的机械折叠和展开轨迹。因此，通过多次拉伸同一分子，我们检测到每个域的独立折叠或域之间难以捉摸的交换步骤。我们发现交换的二聚体可以直接由完全或大部分折叠的单体形成。在这种情况下，通往域交换二聚体的单体之间的相互作用是限速步骤。这种方法是测试具有相关生理功能的蛋白质的不同建议域交换机制的有用策略。